Devices and methods for detecting cells and other analytes
a cell and analyte technology, applied in the field of medical devices, medical diagnostics, cell counting, can solve the problems of guava easycd4 limited improvement, limited application prospects, and limited application prospects of laboratory monitoring tools to determine cd4 counts in resource-limited settings
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example 1
Isolation of CD4+ T Lymphocytes
[0081]Materials
[0082]3-Mercaptopropyl trimethoxysilane was purchased from Gelest (Morrisville, Pa.). Ethanol (200 proof), glass coverslips (35×60 mm, no. 1), hemacytometer and microslide fieldfinder were obtained from Fisher Scientific (Fair Lawn, N.J.). For chamber fabrication, SU-8 photoresist and developer were obtained from MicroChem (Newton, Mass.); silicone elastomer and curing agent were obtained from Dow Corning (Midland, Mich.). Phosphate buffered saline (PBS) was obtained from Mediatech (Herndon, Va.). Lyophilized bovine serum albumin (BSA) was obtained from Aldrich Chemical Co. (Milwaukee, Wis.). The coupling agent GMBS (N-γ-maleimidobutyryloxy succinimide ester) and NeutrAvidin were obtained from Pierce Biotechnology (Rockford, Ill.). Biotinylated mouse anti-human anti-CD4 (clone 13b8.2) was purchased from Beckman Coulter (Somerset, N.J.). Biotinylated mouse antihuman anti-CD36 (clone SMO) was obtained from Ancell (Bayport, Minn.). Alexa Fl...
example 2
Arrangements of Capture Chambers
[0099]A flat microfluidic chamber (FIG. 7) has been used for isolating various target cells from whole blood (Table 1). The principle for specific cell capture is the combination of specific antibodies, well controlled shear stress conditions inside the device, and efficient passivation of the surfaces to prevent nonspecific binding of unwanted cells. Following optimization, the purity of cell capture can be as high as 99% and the yield of capture as high as 80%. One approach for increasing the purity in unfavorable situations relies on the use of isolation chambers in series (FIG. 8), where the first chamber depletes the cells that would otherwise contaminate the sample of interest in the second chamber. The captured cells can be imaged using standard microscope (FIG. 9), counted, and RNA and protein extracted (FIG. 10) for further analysis.
example 3
HIV-Specific CD8 T Cell Isolation
[0100]We developed a PDMS microfluidic device coated with pMHC class I pentamers for the capture of disease-specific CD8+ T cells (FIG. 11). In our experiments, we used soft lithography and SU-8 fabrication techniques to create microchannels within a PDMS mold. The PDMS mold and a silica substrate were then exposed to oxygen plasma treatment before being irreversibly sealed together. The hydroxyl groups on the PDMS and silica surfaces were then treated with 3-mercaptotrimethoxysilane in anhydrous ethanol, resulting in the formation of thiol-terminal groups. After washing off unreacted silane solution with anhydrous ethanol, the PDMS device was flushed with the heterobifunctional crosslinker GMBS in anhydrous ethanol, during which the thiol group on the silane reacts specifically and covalently with the maleimide region of GMBS. This leaves the succinimide residue of the GMBS available for protein attachment, and after flushing unreacted GMBS with 1×P...
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